Generally, there are several methods for milk clotting/gelation, in particular acidification and renneting. Besides, also an enzymatic, covalent crosslinking can induce a gelation of proteins. Therefore several possible applications of transglutaminase in milk products are given. However, an incubation of transglutaminase in milk or milk concentrates even at high enzyme concentrations does not result in a gelation due to the electrostatic repulsion between the casein micelles at neutral pH-values. Therefore, transglutaminase alone is not a sufficient reagent for the preparation of a relatively firm cheese. Only by a reduction of this electrostatic repulsion through a pH-drop or through the effect of rennet at relevant protein concentrations and relevant transglutaminase concentrations a gelation may be achieved.
Amino acids of animal- and plant-based proteins may be crosslinked by enzymes, such as transglutaminase (EC 2.3.2.13) in a known manner. Covalent bonds formed in the enzyme treatment withstand different process conditions, such as heating and mixing, well. From milk proteins, caseins and particularly the κ-casein, are the best substrate for transglutaminase. The β-casein also contains a lot of glutamine and lysine, which are joined together by a transglutaminase enzyme.
In cheese making, transglutaminase is used to increase the cheese yield, with optional approach to coprecipitate whey proteins with casein. In milk, transglutaminase in particular crosslinks casein proteins whereby a network structure is formed, which results in increased yields of a cheese curd. It has been found that heat treatment of milk still enhances the protein crosslinking activity of transglutaminase.
It is known that milk contains substances that inhibit the activity of transglutaminase. These inhibiting substances are deactivated in a heat treatment of milk. On the other hand, it is known that the content of said substances in relation to the total content of proteins and fat is reduced in ultrafiltration of milk.
EP 1057411 A2 discloses a process for incorporating whey proteins into cheese using transglutaminase. Transglutaminase treatment is executed on liquid which is fortified with whey protein. A further liquid containing casein is blended with the transglutaminase-treated liquid. A rennet is then added to provide a cheese curd with a high proportion of whey proteins.
EP 0711504 A1 discloses a process for producing cheese using transglutaminase. Transglutaminase is added to a milk protein solution before, after or simultaneously with the addition of a milk clotting enzyme. It is reported that a cheese curd is produced in a larger amount compared to the conventional methods.
WO 97/01961 discloses a process for making cheese where transglutaminase is added to cheesemilk and incubated for a suitable period. A rennet is then added to provide a coagulate which is further processed into cheese. It is reported that improved yields of cheese are obtained.
Drawback of the above cheese making processes using transglutaminase is that there are processes where transglutaminase remains active, whereby deficiencies in organoleptic properties are arised in resulting cheese products, especially during ripening and a long-term storage. Organoleptic failures such as defects in taste, aroma and texture can be seen in ripened cheese in particular. Also, problems in subsequent coagulation of cheese milk with a rennet can be foreseen in the known cheese making processes.
As stated above, it is known that a cheese yield can be increased by using transglutaminase in cheese making. In particular, transglutaminase increases the amount of caseinomacropeptides in cheese. However, if transglutaminase is added in too large amounts to cheese milk, the subsequent coagulation of the cheese milk with a rennet is inhibited. Moreover, it has been found that transglutaminase remains active during ripening of cheese which makes the ripened cheese tough. Thus, it is highly important that the addition of transglutaminase to cheese milk and the incubation time with transglutaminase are carefully controlled in order to provide cheeses in an efficient manner without reduction of organoleptic properties of the resulting cheese.